Clinical and experimental studies have demonstrated beneficial effects of estrogens on the human cardiovascular system. Recently, however, the Women's Health Initiative study has indicated that combinational postmenopausal hormone replacement therapy may actually increase the risk of coronary artery disease (CAD), whereas studies with unopposed estrogen therapy are still ongoing. Clearly, there is much confusion concerning the physiological and/or therapeutic effects of estrogen. Because CAD is the most common cause of death for both women and men in the United States, a major challenge facing biomedical research is to identify and characterize the molecular basis of estrogen action on the coronary circulation. Helping to meet this challenge is the direct focus of the present proposal. We propose an integrated, comprehensive investigation of how estrogen effects coronary artery smooth muscle cells (CASMC), and thereby influences coronary blood flow. Our preliminary data indicate dual and opposite effects of estrogen on porcine coronary arteries: both relaxation and contraction. Therefore, we believe these data can shed some significant light upon one of the most important controversies in cardiovascular medicine. The hypothesis of the proposal is that estrogen can both contract and relax coronary smooth muscle by targeting a single enzyme: Type 1 or neuronal NOS, in coronary myocytes to release either a vasodilator (NO) or a vasoconstrictor (superoxide) substance to modulate Ca2+ or K+ channel activity. Aim 1 will determine the effects of estrogen on isolated coronary arteries in vitro. Pharmacological studies will identify the signal transduction and ionic mechanisms underlying estrogen-induced contraction of coronary arteries. Aim 2 will investigate the cellular/molecular basis of estrogen's dual effects by employing both whole-cell and single-channel patch-clamp studies to examine the effects of estrogen on calcium and potassium channels directly in single coronary myocytes. Aim 3 will employ both molecular, biochemical, and cellular fluorescence studies to identify the NOS isoform involved in the response to estrogen, and determine the role of superoxide in estrogen-induced contraction. Aim 4 will identify and characterize signaling molecules that link estrogen receptor activation to NOS activity (e.g., HSP90, PI3 kinase, Akt). Co-immunoprecipitation will determine estrogen-stimulated bimolecular interaction of these molecules, and we will employ molecular expression techniques to overexpress and/or knock-out critical signaling molecules. The function of these molecules will be characterized by employing cellular (NO fluorescence) and molecular (patch-clamp) functional studies, and findings will be related back to function of intact coronary arteries. The long-term goal is to understand how estrogen can either contract or relax coronary arteries, and thereby help to reconcile the apparent controversy between basic research into estrogen action and clinical trials. It is hoped that these findings will contribute to the development of new therapeutic measures which will make the potential health benefits of estrogen therapy available to both men and women of all ages.